Cartilage is an avascularized tissue and has been studied as a potential candidate containing anti-angiogenic factors. It is also a tissue which is relatively resistant to tumor development. The tumor associated with cartilage, chondrosarcoma, is the least vascularized of solid tumors. Angiogenesis is one of the important factors in the development of a tumor. Discrete solid tumor masses appear if the tumor cells can provoke the adjacent vascular network to expand in order to supply their nutritional needs. Therefore, the factors involved in the stimulation of angiogenesis have been studied for their role in the development of tumor and anti-angiogenic factors as well as drugs having an angiogenic inhibitory activity have been also investigated as tools for controlling the growth or for effecting regression of tumors.
It has been discovered that scapular cartilage in calves contains a substance that inhibits the vascularization of solid tumors (Langer et al. (1976) Science. 193: 70-72). Because of its encouraging potential as anti-tumor agent, sources of greater supply of cartilage have been looked for.
Sharks are animals being a potential source of this kind of angiogenesis inhibitor because their endoskeleton is composed entirely of cartilage (6% of their body weight versus 0.6% in calves). Sharks have also as an interesting characteristic a low propensity to developing tumors. Many hypotheses have been elaborated to explain this low probability of developing tumors in sharks. It has been shown inter alia that IgM antibodies able to readily attack any aggressing agent, or that macrophages capable of differentiating normal cells from neoplastic cells and of destroying the latter. Rosen and Woodhead (1980) in Medical Hypotheses. 6:441-446 have postulated that the rarity of tumors in elasmobranchs (a group to which pertain sharks and rays) might be due to the high ionic strength of their tissues, which is equivalent to a high body temperature. In these conditions, these authors believe that the immune system exerts a close to 100% immunological surveillance. It has also been discovered that sharks produce an aminosterol having antibacterial and antiprotozoal properties. Finally, Lee and Langer (1983) in Science 221: 1185-1187 and Folkman and Klagsbrun (1987) in Science. 235: 442-447 have shown that sharks produce a substance which inhibits neovascularization. Lee and Langer (op.cit.) have isolated this substance by extracting it from shark cartilage in denaturing conditions (guanidine extraction). This process of extraction is however very long (41 days), and the yield of active components is far from excellent. While the active substance isolated from calves has a molecular weight of about 16 kilodaltons (kDa), the same group of researchers have not given a precise molecular weight to the one retrieved in sharks. This substance is only defined has having a molecular weight higher than 3500 Da. Oikawa et al. (1990) in AA Novel Angiogenic Inhibitor Derived from Japanese Shark Cartilage (I). Extraction and Estimation of Inhibitory Activities Toward Tumor and Embryonic Angiogenesisxe2x89xa1 (Cancer Letters 51: 181-186) have applied the same method of extraction as the one described by Lee and Langer, but of a much shorter duration (2 days instead of 41 days). The anti-angiogenic substance isolated from shark cartilage by Oikawa et al. is restricted to a molecule having a molecular weight ranging from 1000 to 10,000 Da. Schinitsky (U.S. Pat. No. 4,473,551) has described a water extract of crude powdered shark cartilage which fraction of more than 100,000 Da has an anti-inflammatory activity especially in combination with glucosamine. No suggestion of a component of this extract having an anti-angiogenic or anti-tumor activity is made in this patent. Kuetner et al. (U.S. Pat. No. 4,746,729) have isolated a polymorphonuclear neutrophil (PMN) elastase inhibitor from bovine cartilage. This inhibitor has been obtained from an aqueous extract of cartilage from which molecules of a molecular weight of less than 50,000 Da have been retained. Fractionation on Sephacryl S-200 has given numerous fractions from which those of 10-40 kDa have been pooled after they have demonstrated an anti-elastase activity. The active component has an isoelectric point of 9.5 and might have a molecular weight of about 15,000 Da. Kuetner et al. (U.S. Pat. No. 4,042,457) have also shown that bovine cartilage has a component of a molecular weight of less than 50,000 Da which has a cell proliferation inhibitory activity without any activity on endothelial cell growth. Balassa et al. (U.S. Pat. No. 4,822,607) have obtained a cartilage extract in an aqueous solution, which extract has an anti-tumor activity. However, we have observed no anti-angiogenic activity in an extract obtained by reproducing Balassa=s method. Spilburg et al. (U.S. Pat. No. 4,243,582) have isolated two glycoproteins of molecular weight of 65 kDa and of isoelectric point of 3.8 from bovine cartilage (guanidine-extraction) which show anti-trypsin activity and an endothelial cell growth inhibitory activity.
Calf and shark cartilage contain many biological activities such as pro-inflammatory activity, anti-inflammatory activity, anti-angiogenic activity, lysozyme activity, cell growth-promoting activity, inhibitory activity against types I and IV collagenase, elastase, and other proteases like trypsin, chymotrypsin and plasmin. However, nobody has yet obtained a cartilage extract which comprises a pool of clinically valuable activities, and particularly with new activities.
Shark cartilage anti-angiogenic component(s) have been generally tested in rabbit corneal pocket assay or in chick chorioallantoic membrane (CAM) assay. Up to date, whole powdered cartilage has been tested directly on tumors in vivo, on human melanoma xenograft implanted in nude mice (U.S. Pat. No. 5,075,112), as well as tested in CAM tests for its anti-angiogenic effect. Even though an anti-tumor effect has been assigned to cartilage extracts, this effect has most often been attributed to the anti-angiogenic component which deprives the tumor of blood supply. Up to now, there is no evidence in the art that a shark cartilage has a direct effect on tumor cell proliferation.
A few methods of obtaining shark cartilage extracts and fractions are already known. Some of them produce a powdered crude cartilage without any extraction (U.S. Pat. No. 5,075,112). Others use denaturing or chaotropic agents like guanidine (U.S. Pat. No. 4,243,582). Others perform a pre-treatment of cartilage by way of an enzymatic digestion to get rid of any muscular, nervous or vascular structures surrounding the cartilage, which pre-treatment step is followed by the elimination of fats in organic solvents, and then the active components are extracted in an aqueous phase. (Balassa et al. U.S. Pat. Nos. 3,478,146, 4,350,682, 4,656,137 and 4,822,607). The effect of such pre-treatment on the preservation of the integrity of the biologically active cartilage components is not known. If too extensive, an enzyme digestion may hydrolyze active protein components. For example, Balassa=s method (U.S. Pat. No. 4,822,607) produces a liquid extract without anti-angiogenic activity; this lost may be the result of such enzymatic degradation, or else, the anti-angiogenic is masked or antagonized by other molecules. Balassa""s method does not include a fractionation step which would further enrich an extract in active components, and which may remove undesirable molecules. Others simply produce aqueous extracts (in water (U.S. Pat. No. 4,473,551) or salt solutions (U.S. Pat. No. 4,746,729)) of cartilage by eliminating the unsolubilized material. Among the latter, specific fractions of specific molecular weights have been particularly retained for further study and purification (see discussion above). There is no process in the art, that leads to the preparation of a cartilage extract having substantially all the hydrosoluble active components of cartilage.
Moreover, the methods of the prior art are too lengthy to be of a practical purpose and they do not necessarily yield sufficient amounts of active components. Amongst the recovered components, some are not recovered at all or in insufficient yield to show detectable activity or some have been disregarded by focusing on the preparation of one specific component having one activity.
Angiogenesis is not only involved in cancer development. Many diseases or conditions affecting different physiological systems (indicated in parentheses) are angiogenesis-dependent among which the following examples: arthritis and atherosclerotic plaques (bone and ligaments), diabetic retinopathy, neovascular glaucoma, macular degeneration, ocular herpes, trachoma and corneal graft neovascularization (eye), psoriasis, scleroderma, rosacea, hemangioma and hypertrophic scarring (skin), vascular adhesions and angiofibroma (blood system). Therefore, any new and potent anti-angiogenic xe2x80x9cfactorxe2x80x9d could find an use in the treatment of these diseases as well as in cancer therapy and other angiogeno-dependent diseases. Moreover, since many of the above-mentioned diseases and conditions also have an inflammatory etiological component, any new and potent anti-inflammatory xe2x80x9cfactorxe2x80x9d or A componentxe2x89xa1 could find a use in the treatment of these diseases and conditions as well as of any other inflammatory diseases or conditions. Furthermore, since metalloproteases like collagenases are involved in a diversity of diseases and conditions like cancer, inflammation and premature aging of the skin (collagen degrading activity), a new and potent anti-collagenolytic xe2x80x9cfactorxe2x80x9d could find a use in the treatment of diseases or conditions having a collagenolytic or a matrix metalloprotease etiological component. Because angiogenesis, inflammation and proteolysis may be encountered alone or in combination in a large variety of diseases or conditions, a product capable of antagonizing at least all these activities without affecting normal body functions would be of a great therapeutic value. Furthermore, a product which would have a direct anti-tumor activity would also have a significant therapeutic value.
The present invention provides a new method of producing cartilage extracts which has the advantage of containing a plurality of therapeutically valuable activities. Amongst those, anti-angiogenic, anti-inflammatory, anti-collagenolytic, in vivo anti-tumor proliferating and direct in vitro anti-tumor proliferating activities have been confirmed to be present in satisfying concentrations in a cartilage extract obtained from shark. All activities have been obtained in a liquid extract of shark cartilage, and some of them have been obtained or verified as being present in a solid extract of the same.
The present invention relates to a new method of preparing a fractionated cartilage extract comprising water soluble biologically active components, the majority of which have a molecular weight of less than about 500 kDa, the method comprising the step of:
first fractionating a crude cartilage extract comprising water soluble biologically active components obtained from cartilage material such that a major portion of the biologically active components having a molecular weight of greater than about 500 kDa are separated from a major portion of biologically active components having a molecular weight of less than about 500 kDa to form a first fractionated cartilage extract.
A first fractionated cartilage extract has been obtained from shark cartilage and is referred to hereinbelow as a A cartilage extractxe2x89xa1. The term A fractionatedxe2x89xa1 will be omitted, even though the cartilage extract has been fractionated to remove molecules heavier than about 500 kDa.
This method has the advantage of being easy to perform and efficient. High yields of cartilage extract have been obtained, which extract, particularly obtained from fresh or frozen/thawed shark cartilage, contains at least all the above-mentioned biological activities. Other sources of cartilage can be used in this process. It is preferably performed at cold, cool or ambient temperatures (about 0 to 20 EC. although the biologically active components can withstand temperatures as high as about 401 C.), in non-denaturing conditions (preferably, the extraction medium is an aqueous solution or pure water), at a near neutral pH (about 5 to 8) to maximize the probability of recovering compounds of a priori unknown physico-chemical characteristics. According to this process, cartilage components can be extracted in a low volume of solution (as low as 1 L for 1 Kg of cartilage). Homogenization of cartilage may be performed for a short period of time (as short as 10 to 20 minutes). Homogenization and extraction in the extraction medium results in the formation of particles and a crude extract which are separated by mechanical and physical means. The crude extract is then fractionated to remove molecules having a molecular weight higher than 500 kDa. A liquid extract is the fractionated extract comprising water soluble biologically active components having a molecular weight less than about 500 kDa. In one embodiment, the solid extract comprises the insoluble particles separated from the liquid extract. In another embodiment, the solid extract is a dried form of the liquid extract. Homogenization reduces the size of cartilage particles and thereby maximizes the extractable surface area of the cartilage. Other known ways of reducing the particle size of solids, in particular cartilage, may be used, as long as a major portion of the biological activities being extracted are preserved and remain extractable.
This invention relates to cartilage extracts, particularly to extracts from elasmobranch species, more particularly shark. The solid extract has shown activity. It may contain collagen and non-hydrosoluble components. It may also contain a residual activity of what was extracted in the cartilage extract. The cartilage extract is very rich in activities. It can be used as such or it can be concentrated. A concentration step which favors the maintenance of biological activities is conducted. Ultrafiltration on a membrane having a nominal molecular weight cut-off value of about 1 kDa has been used to concentrate the cartilage extract of this invention. Nanofiltration on a membrane having a nominal molecular weight cut-off value of about 100 Da should even be better to concentrate the biological activities of the cartilage extract, preventing the loss of any activity assigned to very small molecules. Finally, concentration by evaporation or lyophilization can also be performed, in so far as such treatment does not substantially reduce the activity of the extract. Stabilizers may be used to protect the cartilage extract to minimize loss of activity during lyophilization.
The cartilage extract ( less than 500 kDa) has been further fractionated to characterize the active components thereof. Active fractions have been obtained by performing additional fractionation steps. Some fractions tested for their anti-tumor activity on tumor cell lines have been grossly characterized by their molecular weight and isoelectric point. Others have been assigned an activity, particularly anti-collagenolytic, anti-metalloproteasic or anti-angiogenic. Therefore, valuable activities are recovered in cartilage extract and fractions thereof, which may be advantageously used. In lieu of administering high amounts of powdered cartilage, a more acceptable and enriched extract may now be administered.
The present invention also relates to any therapeutic or cosmetic compositions comprising as an active ingredient an effective amount of a concentrated or dilute cartilage extract preferably obtained from shark. These compositions are generally used in dermatological or cosmetic formulations due to the observed activities of the cartilage extract. In this respect, the observed anti-angiogenic, anti-metalloproteasic and anti-inflammatory activities, and the antagonistic effect of cellular differentiation mediated by the protein kinase C signal transduction pathways in keratinocytes, and the antagonistic effect on VEGF (vascular endothelium growth factor) activity, are all considered as possible mechanisms upon which new uses of the shark cartilage extract can be developed.
The invention also provides a method of treating a variety of mammalian skin diseases or disorders including, for example, Reiter""s syndrome, pityriasis rosea, lichen planus, pityriasis rubra pilaris, secondary syphilis, mycosis fungoides, ichthyosiform eruptions, sclerodermia, hyperthrophic scar, papulosquamous disease, psoriasis, rosacea, eczema and acne. Preferred embodiments of the invention provide the following:
1) a method for inhibiting angiogenesis in mammalian skin;
2) a method for reducing telangiectesia in mammalian skin;
3) a method for reducing spider veins or varicose veins in mammalian skin;
4) a method for inhibiting endothelial cell proliferation in mammalian skin;
5) a method for treating cancer in mammalian skin, particularly a melanoma;
6) a method for decreasing the expression of rosacea in mammalian skin;
7) a method for reducing papulosquamous skin disease in mammalian;
8) a method for reducing the appearance of peri-orbital dark circles in mammalian;
9) a method for reducing inflammation in mammalian, be it caused by a chemical irritant, a physical abrasion, U.V. radiation, an allergen or an infectious agent.
10) a method for soothing irritated skin in mammalian;
11) a method for inhibiting activated-keratinocyte differentiation in mammalian skin;
12) a method for reducing acne in mammalian skin;
13) a method for decreasing the expression of eczema in mammalian skin;
14) a method for inhibiting metalloprotease activity in mammalian skin;
15) a method for treating warts in mammalian;
16) a method for enhancing wound repair in mammalian;
17) a method for enhancing skin barrier function in mammalian;
18) a method for regulating wrinkles and atrophy in mammalian skin; and
19) a method for retarding premature aging in mammalian skin.
A plurality of diseases or disorders having simple or complex etiologies will benefit from treatment with the present cartilage extract. Other medical fields of use, including but not limited to dermatological use, are within the scope of this invention. Dermatological diseases or disorders that have up to date been difficult to treat with actual short and long term therapies because of their complex etiology, are susceptible to treatment with the cartilage extract of this invention. Furthermore, since the cartilage extract has been successfully tested in a variety of cancers, arthritis, psoriasis, rosacea and acne cases, compositions and methods for treating diseases, conditions or disorders having one or more etiological components selected from tumor proliferation, angiogenesis, inflammation and metalloprotease activity such as collagenolysis, are within the scope of this invention, without any limitation as to the dosage form, the route of administration way and the tissue to be treated.